The nature of the forces involved in ligand binding by antibodies and substrate binding by enzymes is similar, viz., hydrogen bonding, electrostatic interaction and hydrophobic effect. The energy obtained from enzyme-substrate binding may be visualized to force electronic strain in the substrate and facilitate the formation of a transition state. There is strong evidence for the theory that enzymes bind the transition state of the reaction they catalyze better than the ground state, resulting in a reduced free energy of activation for the reaction (1). This has come to be known as the transition state theory of enzymatic catalysis. Other factors that may facilitate enzymatic catalysis are the proximity and orientation effects--apposition of correctly oriented reactants within the active site of the enzyme would reduce the requirement for a large number of random collisions prior to a productive reactant interaction. In principle, antibodies could catalyze chemical reactions by similar means.
The first report of chemical conversion of a ligand by an antibody appeared in 1980 (2), but the steroid ester hydrolysis by a rabbit polyclonal antiserum described in this report was stoichiometric rather than catalytic. Subsequently, antibodies have been demonstrated to catalyze or facilitate chemical reactions, including acyl transfer (3), pericyclic (4) and redox reactions (5). It is generally believed that these antibodies obtain their catalytic properties, like enzymes, from their ability to bind the transition state of the ligand better than its ground state.
The known catalytic antibodies include those generated by immunization with selected epitopes as described above, and those which have been shown to occur naturally (6). These naturally occurring antibodies are produced by an animal's immune system to the animal's own cellular component (self-antigen), hence "autoantibodies", as opposed to antibodies elicited with an antigen introduced by specific immunization against a target antigen, and are known to enhance the rate of a chemical reaction, e.g., the cleavage of a peptide bond.
Antibodies with enzymatic activity offer the possibility of specific, high efficiency catalytic chemical conversion of ligands. Many biological mediators are peptides or proteins, including the antigens of pathogenic organisms, hormones, neurotransmitters and tumor specific antigens. It should be possible to utilize the vast repertoire of specificities that the immune system encompasses to catalyze chemical reactions not within the scope of naturally occurring enzymes. The combination of antibody specificity with the catalytic power of enzymes has the potential of generating potent therapeutic agents, e.g., catalytic antibodies capable of specifically hydrolyzing key viral coat proteins, tumor specific proteins, or endogenous proteins involved in disease. The utilization of these catalytic antibodies in medicine and industry would be greatly enhanced if a specific, selective method of inhibiting the activity of these antibodies were also available.
For example, such inhibition could be useful in the treatment of autoimmune diseases. It is well known that certain autoimmune diseases are associated with autoantibodies directed against hormones and cell surface antigens. Examples of these diseases and associated autoantibodies are:
______________________________________ Disease Autoantibody to ______________________________________ Diabetes Insulin, Insulin receptor Myasthenia gravis acetylcholine receptor Graves disease thyroid stimulating hormone receptor Systemic lupus erythematosus small nuclear RNA, DNA, histones Pernicious anemia Intrinsic factor of Castle, gastric parietal cell antibodies ______________________________________
It is known to treat autoimmune diseases generally by means of non-specific anti-immune treatments. These known treatments include steroids, alkylating agents, radiation, plasmaphoresis, and surgical removal of the spleen. Each of these treatments suffers from many disadvantages well known in the art such as impairment of the patient's immune system. Since catalytic autoantibodies are likely to cause more harm than non-catalytic antibodies, it is now believed that the autoimmune diseases are caused by catalytic autoantibodies directed against nucleic acids, key regulatory peptides and proteins (e.g., insulin, glucagon, prolactin, VIP, substance P, blood clotting factors) and the cell surface receptors for these agents.
For example, asthma is believed to be caused by a deficiency of vasoactive intestinal peptide (VIP). VIP is a 28 amino acid peptide originally isolated from the intestine but now recognized to be a neuropeptide widely distributed in the central and peripheral nervous systems. There is evidence that VIP is a neurotransmitter in its own right. In addition, VIP may modulate neuroetransmission by classical transmitters and has been implicated in regulation of blood pressure, bronchial tone, neuroendocrine activity and exocrine secretion. VIP appears to be the major neurobronchodilator in humans and a diminished influence of VIP on the airways may permit a dominance of constrictor influences, and may underlie airway hyperactivity in asthma.
VIP belongs to a family of structurally related peptides, other prominent members of which are peptide histidine isoleucine (PHI), growth hormone releasing factor (GRF) and secretin. Like the peptides themselves, there is evidence that the receptors for VIP, GRF, PHI and secretin are related. Receptors for VIP are found in lung, vascular smooth muscle, brain, pancreas, skin, intestine and other tissues. The amino acid sequence of VIP is as follows:
H S D A V F T D N Y T R L R K Q M A V K Y L N S I L N-NH.sub.2.
It has been discovered that VIP binding antibodies exist in human circulation (7-9). Immunoprecipitation with anti-human IgG as well as chromatography on DEAE-cellulose, gel filtration columns and immobilized protein-G indicate that the plasma VIP binding activity is largely due to IgG antibodies. The antibodies to VIP are present in the blood of 18% of asthma patients and 30% of healthy subjects with a history of habitual muscular exercise, compared to only 2% of healthy subjects with no such history. The antibodies are highly specific for VIP, judged by their poor reaction with peptides related to VIP (i.e., GRF, PHI and secretin). A clear difference in the VIP binding affinity of the antibodies from asthma patients (mean K.sub.bind =0.13 Nm.sup.-1) and healthy subjects (mean K.sub.bind =7.7 Nm.sup.-1) was observed--the antibodies from the asthmatics exhibiting a 60-fold greater binding affinity. The immune IgG from asthma patients reduces the binding of VIP by lung receptors as well as the VIP-responsive synthesis of cyclic AMP in lung membranes. Thus, the antibodies can be directed against an epitope(s) that binds the receptor or maintains the receptor-binding epitope in an active conformation.
These antibodies are detected by measuring their binding to porcine .sup.125 I-VIP. Human and porcine VIP are structurally identical (10). Thus, the porcine VIP-reactive antibodies found in asthma patients are autoantibodies. It had been observed that diabetics positive for plasma VIP-antibodies had been treated with insulin contaminated with VIP, suggesting that the formation of antibodies was related to the VIP contaminant (11).
The antigenic stimulus leading to formation of these autoantibodies cannot be identified with certainty. Candidate stimuli include exposure to viral determinants similar in sequence to VIP[e.g., Peptide-T, an epitope found on the human immunodeficiency virus (12)] and dietary ingestion of avian, fish and turtle VIP known to be structurally different from human VIP (13, 14). Muscular exercise, which results in increased plasma VIP immunoreactivity (15, 16), could also be a potential stimulus for VIP autoantibody formation. Indeed, asthma and muscular exercise appear to be associated with an increased incidence of autoantibodies directed against VIP.
Irrespective of the type of antigenic stimulation leading to VIP-autoantibody formation, these antibodies may produce important biologic changes. The range of K.sub.a values observed for the autoantibodies of asthma patients is similar to that reported for VIP receptors present in the lung and other tissues (17, 18), and these antibodies neutralize VIP receptor binding. It is possible that VIP-autoantibodies found in asthmatics neutralize the effect of VIP in the airways.
It has been discovered that these VIP autoantibodies catalyze the hydrolysis of VIP between amino acid residues 16 and 17, i.e. between glutamine (Gln.sup.16) and methionine (Met.sup.17). These are described in U.S. application Ser. No. 342,081, filed Apr. 25, 1989, the disclosure of which is incorporated herein by reference.
Thus, specific inhibitors of catalytic autoantibodies, e.g., an inhibitor of the autoantibody which catalyzes the cleavage of VIP, would provide an important therapeutic advance for the treatment of catalytic autoantibody autoimmune disease, in particular asthma and similar respiratory diseases. More generally, inhibitors of catalytic antibodies would provide the art with the means to tailor and control the catalytic activity of such antibodies, regardless of how they are used.